CN119365216A - A pharmaceutical composition containing an antibody-drug conjugate and its use - Google Patents

Abstract

A pharmaceutical composition containing an antibody-drug conjugate and its use. Specifically, it relates to a pharmaceutical composition, which contains an antibody-drug conjugate in a buffer. The pharmaceutical composition has good stability.

Description

Pharmaceutical composition containing antibody drug conjugate and application thereof Technical Field

The present disclosure is in the field of pharmaceutical formulations, and in particular relates to a pharmaceutical composition comprising an antibody drug conjugate and its use as an anticancer drug.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

The antibody drug conjugate (antibody drug conjugate, ADC) connects monoclonal antibody or antibody fragment with cytotoxin with biological activity through stable chemical linker compound, makes full use of the specificity of antibody to tumor cell surface antigen combination and cytotoxin high efficiency, and simultaneously avoids the defects of low curative effect and overlarge toxic and side effect of the former. This means that the antibody drug conjugate can bind tumor cells precisely and reduce the effect on normal cells compared with conventional chemotherapeutics (Mullard A,(2013)Na-ture Reviews Drug Discovery,12:329–332;DiJoseph JF,Armellino DC,(2004)Blood,103:1807-1814).

There are several classes of small molecules with cytotoxicity for antibody drug conjugates, one of which is camptothecin derivatives, which have antitumor effects by inhibiting topoisomerase I. The reported camptothecin derivative, isatecan (chemical name: 1S, 9S) -1-amino-9-ethyl-5-fluoro-2, 3-dihydro-9-hydroxy-4-methyl-1H, 12H-benzo [ de ] pyrano [3 ^,,4^,:6, 7] imidazo [1,2-b ] quinoline-10, 13 (9H, 15H) -dione), was used in the literature of Antibody Drug Conjugates (ADC) as WO2014057687; CLINICAL CANCER RESEARCH (2016) 22 (20): 5097-5108; cancer Sci (2016) 107:1039-1046. There is still a need to further develop ADC drugs with better therapeutic effects.

However, ADCs have more complex heterostructures than antibodies and therefore present a greater challenge for ADC formulations for therapeutic purposes.

Disclosure of Invention

The present disclosure provides a pharmaceutical composition comprising an antibody drug conjugate and a buffer, wherein the antibody drug conjugate has the structure shown below:

antibody drug conjugates as described above refer to the preparation of antibody conjugate example 9FADC-2 on pages 45-47 of the description of WO2020063673A 1;

Wherein:

H1702DS is an anti-B7H 3 antibody, the source of which is referred to the antibody H1702DS on page 44 in WO2020063673A1, comprising a heavy chain as shown in SEQ ID NO. 1 (SEQ ID NO. 14 in WO2020063673A 1) and a light chain as shown in SEQ ID NO. 2 (SEQ ID NO. 16 in WO2020063673A 1):

h1702DS heavy chain amino acid sequence (SEQ ID NO: 1)

H1702DS light chain amino acid sequence (SEQ ID NO: 2)

N is 1 to 10, preferably 1 to 8, more preferably 3 to 5, more preferably about 4;

The buffer of the pharmaceutical composition is a succinate buffer, a histidine buffer, preferably a succinic acid-succinic acid sodium salt, histidine-acetate or histidine-hydrochloride buffer.

The buffer concentration of the pharmaceutical composition is from about 15mM to about 50mM, preferably from about 20mM to about 40mM, more preferably about 30mM.

The pharmaceutical composition has a pH of about 4.5 to about 6.5, preferably a pH of about 5.0 to about 6.0, more preferably a pH of about 5.5 to about 5.6.

In alternative embodiments, the pH of the buffer in the pharmaceutical composition is about 4.5 to about 6.5, non-limiting examples include about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, and any range between these point values, preferably about 5.0 to about 6.0, more preferably about 5.5 to about 5.6.

In an alternative embodiment, the succinate buffer in the pharmaceutical composition is succinic acid-succinic acid sodium salt, and the histidine salt buffer is preferably histidine-acetate or histidine-hydrochloride buffer.

In an alternative embodiment, the pharmaceutical composition further comprises a surfactant. Can be selected from polysorbate, polysorbate 20, polysorbate 80, polyhydroxyene, triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocamidopropyl-betaine, oleamidopropyl-betaine, myristamidopropyl-betaine, palmitoamidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmamidopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoa acyl, sodium methyl taurate, polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol, and the like. The preferred surfactant is polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

In alternative embodiments, the concentration of surfactant in the pharmaceutical composition is from about 0.01mg/mL to about 1.0mg/mL, preferably from about 0.05mg/mL to about 0.5mg/mL, more preferably from about 0.1mg/mL to about 0.4mg/mL, or from about 0.2mg/mL to about 0.3mg/mL, and most preferably about 0.2mg/mL, non-limiting examples include 0.02mg/mL、0.05mg/mL、0.1mg/mL、0.15mg/mL、0.2mg/mL、0.25mg/mL、0.3mg/mL、0.35mg/mL、0.4mg/mL、0.45mg/mL、0.5mg/mL、0.8mg/mL, and any range between these point values.

In an alternative embodiment, the aforementioned pharmaceutical composition further comprises a sugar. The "sugar" of the present disclosure includes conventional compositions (CH ₂O)_n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like, the sugar may be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, stachyose, maltose, lactulose, maltotriose, sorbitol, maltitol, lactitol, iso-maltotriose, and the like.

In alternative embodiments, the concentration of sugar in the aforementioned pharmaceutical compositions is from about 25mg/mL to about 80mg/mL, preferably from about 30mg/mL to about 60mg/mL, preferably about 40mg/mL, non-limiting examples include 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, 55mg/mL, 60mg/mL, 65mg/mL, 70mg/mL, 75mg/mL, 80mg/mL, and any range between these spot values, preferably 40mg/mL.

In an alternative embodiment, the aforementioned pharmaceutical composition further comprises an amino acid and salts thereof, preferably selected from glycine and arginine hydrochloride, more preferably glycine.

In alternative embodiments, the glycine concentration in the aforementioned pharmaceutical compositions is from about 5mg/mL to about 10mg/mL, from about 5.3mg/mL to about 9.8mg/mL, from about 6mg/mL to about 9mg/mL, preferably from about 7mg/mL to about 8mg/mL, non-limiting examples include 6mg/mL, 6.5mg/mL, 7mg/mL, 7.2mg/mL, 7.6mg/mL, 7.8mg/mL, 8mg/mL, 8.5mg/mL, 9mg/mL, and any range between these values, most preferably about 7.6mg/mL.

In alternative embodiments, the aforementioned pharmaceutical compositions further comprise arginine hydrochloride at a concentration of about 40mM to about 80mM, preferably about 50mM to about 70mM, non-limiting examples include 40mM, 45mM, 50mM, 55mM, 58mM, 60mM, 65mM, 70mM, 75mM, and 80mM, most preferably about 58mM.

In alternative embodiments, the concentration of the antibody drug conjugate in the pharmaceutical composition is from about 1mg/mL to about 100mg/mL, with non-limiting examples including 1mg/mL、10mg/mL、11mg/mL、12mg/mL、13mg/mL、14mg/mL、15mg/mL、16mg/mL、17mg/mL、18mg/mL、19mg/mL、20mg/mL、21mg/mL、22mg/mL、23mg/mL、24mg/mL、25mg/mL、26mg/mL、27mg/mL、28mg/mL、29mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、90mg/mL、100mg/mL and any range between these spot values, preferably from about 10mg/mL to about 70mg/mL, more preferably from about 20mg/mL to about 50mg/mL, even more preferably from about 20mg/mL to about 30mg/mL or from about 30mg/mL to about 50mg/mL, and even more preferably from about 20mg/mL or about 30mg/mL or about 50mg/mL, as measured by protein concentration. In particular, non-limiting embodiments include 50.1mg/mL、50.2mg/mL、50.3mg/mL、50.4mg/mL、50.5mg/mL、50.6mg/mL、50.7mg/mL、50.8mg/mL、50.81mg/mL、50.82mg/mL、50.83mg/mL、50.84mg/mL、50.85mg/mL、50.86mg/mL、50.87mg/mL、50.88mg/mL、50.89mg/mL、50.9mg/mL、50.9mg/mL、50.91mg/mL、50.92mg/mL、50.93mg/mL、50.94mg/mL、50.95mg/mL、50.96mg/mL、50.97mg/mL、50.98mg/mL、50.99mg/mL、51mg/mL and any range between these point values. The term protein concentration refers to the concentration of the antibody moiety in the antibody drug conjugate.

In alternative embodiments, the buffer concentration in the pharmaceutical composition is from about 15mM to about 50mM, non-limiting examples include 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 30mM, 40mM, 50mM, and any range between these spot values, preferably from about 15mM to about 50mM, more preferably from about 20mM to about 40mM, and most preferably about 30mM.

In alternative embodiments, the range of drug loading (DAR) may be an average number of cytotoxic drugs bound per antibody h1702DS, with non-limiting examples of average numbers of cytotoxic drugs bound per antibody h1702DS of about 1,2, 3, 4, 5, 6,7,8,9,10, 11, 12 and any range between these spot values. Preferably about 3 to 8, about 4 to 8, about 5 to 7, about 4 to 6, more preferably about 3 to 5, most preferably 4.1 cytotoxic agents are combined. n is a decimal or integer.

In an alternative embodiment, the drug loading (DAR) is about 4.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) about 10mg/mL to about 70mg/mL of the antibody drug conjugate, (b) about 0.1mg/mL to about 0.8mg/mL of polysorbate, (c) about 25mg/mL to about 80mg/mL of sugar, (d) about 5mg/mL to about 10mg/mL glycine, and (e) about 15mM to about 50mM succinate buffer, the composition having a pH of about 5.0-6.0;

Preferably, the pharmaceutical composition comprises the following components:

(a) about 20mg/mL to about 70mg/mL of the antibody drug conjugate, (b) about 0.2mg/mL to about 0.8mg/mL of polysorbate, (c) about 25mg/mL to about 80mg/mL of sugar, (d) about 7mg/mL to about 8mg/mL glycine, and (e) about 10mM to about 40mM succinate buffer, the composition having a pH of about 5.0-6.0;

more preferably, the pharmaceutical composition comprises the following components:

(a) about 20mg/mL to about 50mg/mL of the antibody drug conjugate, (b) about 0.2mg/mL to about 0.8mg/mL of polysorbate 80, (c) about 40mg/mL to about 80mg/mL of sucrose, (d) about 7mg/mL to about 8mg/mL of glycine, and (e) about 20mM to about 40mM of succinic acid-succinic acid sodium salt, the pH of the composition being about 5.0-6.0;

Further preferably, the pharmaceutical composition comprises the following components:

(a) about 20mg/mL to about 30mg/mL of the antibody drug conjugate, (b) about 0.2mg/mL polysorbate 80, (c) about 40mg/mL sucrose, (d) about 7.6mg/mL glycine and (e) about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being about 5.5-5.6;

(a) about 30mg/mL to about 50mg/mL of the antibody drug conjugate, (b) about 0.2mg/mL polysorbate 80, (c) about 40mg/mL sucrose, (d) about 7.6mg/mL glycine and (e) about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being about 5.5-5.6;

still further preferably, the pharmaceutical composition comprises the following components:

(a) about 20mg/mL of the antibody drug conjugate, (b) about 0.2mg/mL of polysorbate 80, (c) about 40mg/mL of sucrose, (d) about 7.6mg/mL glycine and (e) about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being about 5.5-5.6.

(A) about 30mg/mL of the antibody drug conjugate, (b) about 0.2mg/mL of polysorbate 80, (c) about 40mg/mL of sucrose, (d) about 7.6mg/mL glycine and (e) about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being about 5.5-5.6.

(A) about 50mg/mL of the antibody drug conjugate, (b) about 0.2mg/mL of polysorbate 80, (c) about 40mg/mL of sucrose, (d) about 7.6mg/mL glycine and (e) about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being about 5.5-5.6.

In an alternative embodiment, the pharmaceutical composition of any of the foregoing is a liquid formulation.

The present disclosure also provides a lyophilized formulation comprising an antibody drug conjugate, characterized in that the formulation, upon reconstitution, forms a pharmaceutical composition as described above.

The present disclosure also provides a method of preparing a lyophilized formulation comprising an antibody drug conjugate comprising the step of lyophilizing the pharmaceutical composition as described above.

In an alternative embodiment, the method of preparing a lyophilized formulation comprising an antibody drug conjugate wherein the lyophilization comprises the steps of prefreezing, primary drying and secondary drying in that order. Lyophilization is performed by freezing the formulation and subsequently sublimating the water at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic or collapse temperature of the formulation. Typically, the temperature of the primary drying is in the range of about-30 to 25 ℃ (assuming the product remains frozen during the primary drying process). The size and type of formulation, the container (e.g., glass vial) containing the sample, and the volume of liquid determine the time required for drying, which may range from a few hours to a few days (e.g., 40-60 hours). The secondary drying stage may be carried out at about 0-40 ℃, depending primarily on the type and size of the container and the type of protein employed. The secondary drying time is determined by the desired residual moisture level in the product and typically takes at least about 5 hours. Typically, the water content of the lyophilized formulation is less than about 5%, preferably less than about 3%. The pressure may be the same as the pressure applied in the primary drying step, preferably the pressure of the secondary drying is lower than the primary drying. The lyophilization conditions may vary with formulation and vial size.

In an alternative embodiment of the present disclosure, 5mL of the composition stock solution is lyophilized, the lyophilization procedure is shown as 5 ℃ and-45 ℃ in sequence, the primary drying temperature is-20 ℃, the vacuum is 10Pa, the secondary drying temperature is 25 ℃, and the vacuum is 10Pa and 1Pa in sequence.

In some embodiments, the lyophilized formulation is stable at 2-8 ℃ for at least 16 days, at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 40 ℃ for at least 7 days, at least 14 days, at least 28 days, or at least 30 days.

The present disclosure also provides a lyophilized formulation comprising an antibody drug conjugate, obtained by freeze-drying a pharmaceutical composition of an antibody drug conjugate as described above.

The present disclosure also provides a reconstitution solution of an antibody-containing drug conjugate, characterized in that the reconstitution solution is obtained by reconstitution of a lyophilized formulation as described above.

In an alternative embodiment, the redissolved solution comprises:

(a) about 10mg/mL to about 70mg/mL of the antibody drug conjugate, (b) about 0.1mg/mL to about 0.8mg/mL of polysorbate, (c) about 25mg/mL to about 80mg/mL of sugar, (d) about 5mg/mL to about 10mg/mL glycine, and (e) about 15mM to about 50mM succinate buffer, the reconstitution solution having a pH of about 5.0-6.0;

preferably, the redissolved solution comprises the following components:

(a) about 20mg/mL to about 70mg/mL of the antibody drug conjugate, (b) about 0.2mg/mL to about 0.8mg/mL of polysorbate, (c) about 25mg/mL to about 80mg/mL of sugar, (d) about 7mg/mL to about 8mg/mL glycine, and (e) about 10mM to about 40mM succinate buffer, the pH of the composition being about 5.0-6.0;

More preferably, the redissolved solution comprises the following components:

(a) about 20mg/mL to about 50mg/mL of the antibody drug conjugate, (b) about 0.2mg/mL to about 0.8mg/mL of polysorbate 80, (c) about 40mg/mL to about 60mg/mL of sucrose, (d) about 7mg/mL to about 8mg/mL of glycine, and (e) about 20mM to about 40mM of succinic acid-succinic acid sodium salt, the reconstitution solution having a pH of about 5.0-6.0;

further preferably, the redissolved solution comprises the following components:

(a) about 20mg/mL to about 30mg/mL of the antibody drug conjugate, (b) about 0.2mg/mL of polysorbate 80, (c) about 40mg/mL of sucrose, (d) about 7.6mg/mL of glycine and (e) about 30mM of succinic acid-succinic acid sodium salt, the pH of the composition being about 5.5-5.6, or

(A) about 30mg/mL to about 50mg/mL of the antibody drug conjugate, (b) about 0.2mg/mL polysorbate 80, (c) about 40mg/mL sucrose, (d) about 7.6mg/mL glycine and (e) about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being about 5.5-5.6.

Still more preferably, the redissolved solution comprises the following components:

(a) about 20mg/mL of the antibody drug conjugate, (b) about 0.2mg/mL of polysorbate 80, (c) about 40mg/mL of sucrose, (d) about 7.6mg/mL glycine and (e) about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being about 5.5-5.6;

(a) about 30mg/mL of the antibody drug conjugate, (b) about 0.2mg/mL of polysorbate 80, (c) about 40mg/mL of sucrose, (d) about 7.6mg/mL glycine and (e) about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being about 5.5-5.6;

(a) about 50mg/mL of the antibody drug conjugate, (b) about 0.2mg/mL of polysorbate 80, (c) about 40mg/mL of sucrose, (d) about 7.6mg/mL glycine and (e) about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being about 5.5-5.6.

The present disclosure also provides a method of preparing the above reconstituted solution comprising the step of reconstituting the aforementioned lyophilized formulation using a solution selected from, but not limited to, water for injection, physiological saline or dextrose solution.

The present disclosure also provides an article of manufacture comprising a container containing a pharmaceutical composition, lyophilized formulation, or reconstituted solution as described above. In some embodiments, the container is a neutral borosilicate glass tube injection bottle.

The disclosure also provides the use of the aforementioned pharmaceutical composition or lyophilized formulation or reconstituted solution or product in the manufacture of a medicament for treating or preventing a tumor.

The present disclosure also provides methods of treating a disease comprising providing a pharmaceutical composition or lyophilized formulation or reconstituted solution or article of manufacture as described previously.

The present disclosure also provides the aforementioned pharmaceutical composition, or lyophilized formulation, or reconstituted solution, or article of manufacture as a medicament, preferably for the treatment or prevention of neoplastic disease.

In alternative embodiments, the disease or tumor is a cancer associated with HER2, HER3, B7H3, or EGFR expression.

In alternative embodiments, the cancer is selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urinary tract cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma.

As is well known to those of skill in the art, one, some, or all of the features of the various embodiments described in the present disclosure may be further combined to form other embodiments of the present disclosure. The above embodiments of the present disclosure and other embodiments resulting from the combination are further illustrated by the following detailed description.

For formulation development of ADC, the inter-chain disulfide bonds opened during the coupling process are oxidized even at low concentration, and the strong hydrophobic toxin coupled affects the stability of the antibody to some extent. If the concentration is increased, the effect tends to be aggravated, so that the ADC is more prone to aggregation/degradation, and cannot be stably present in a low concentration formulation. By reasonable prescription design and screening, a proper high-concentration prescription is screened out, and the stability of the ADC is maintained. The high-concentration ADC preparation of the invention has a larger safe treatment window in clinic, greatly shortens the production time, reduces the consumable cost (penicillin bottle, rubber plug, aluminum cover and the like) and improves the convenience of clinical administration of medical staff.

Detailed Description

The present disclosure provides a pharmaceutical composition that is more advantageous for production and administration, and that is stable in performance. Among other things, the undesirable instability can include any one or more of aggregation, deamidation (e.g., asn deamidation), oxidation (e.g., met oxidation), isomerization (e.g., asp isomerization), clipping (clipping)/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, dissociation of toxins, and the like. In particular, the pharmaceutical compositions described in the present disclosure comprise an antibody drug conjugate and a buffer.

Terminology

For easier understanding of the present disclosure, certain technical and scientific terms are specifically defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

The present disclosure incorporates the entire content of application WO2020063673 into the present application.

An "antibody drug conjugate (antibody drug conjugate, ADC)" is a combination of an antibody via a linker unit with a biologically active cytotoxin or a small molecule drug with cell killing activity.

"Drug loading" is also known as Drug-to-Antibody Ratio (DAR), the average number of drugs conjugated per Antibody in an ADC. It may be in the range of, for example, about 1 to about 10 drugs per antibody conjugate, and in certain embodiments, in the range of about 1 to about 8 drugs per antibody conjugate, preferably in the range of 2-8,2-7,2-6,2-5,2-4, 3-5,5-6,5-7,5-8, and 6-8. By way of example only, and not by way of limitation, the drug loading rate can be 1,2,3,4,5,6,7,8,9, 10. The ADC formulas of the present disclosure include a collection of antibodies conjugated to a drug within the aforementioned ranges. In embodiments of the present disclosure, the drug loading may be expressed as n. The drug loading can be determined by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assays and HPLC.

The term "linker unit" or "connecting fragment" or "connecting unit" refers to a chemical structural fragment or bond that is linked at one end to an antibody or antigen binding fragment thereof and at the other end to a drug, or can be linked to other linkers before being linked to a drug.

The linker, including the extension, spacer and amino acid units, may be synthesized by methods known in the art, such as described in US 2005-0238499 A1. The linker may be a "cleavable linker" that facilitates release of the drug in the cell. For example, acid labile linkers (e.g., hydrazones), protease-sensitive (e.g., peptidase-sensitive) linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al CANCER RESEARCH 52:127-131 (1992); U.S. Pat. No.5,208,020) may be used.

The loading of the cytotoxic drug may be controlled by the following non-limiting methods, including:

(1) The molar ratio of the connecting reagent to the monoclonal antibody is controlled,

(2) The reaction time and the temperature are controlled,

(3) Different reagents are selected.

The amino acid three-letter codes and one-letter codes used in the present disclosure are as described in J.biol. Chem,243, p3558 (1968).

The "antibodies" described in the present disclosure are used in the broadest sense and encompass a variety of antibody structures, including but not limited to full length antibodies and antibody fragments (or antigen binding fragments, or antigen binding portions), so long as they exhibit the desired antigen binding activity. Typically, a natural intact antibody has a tetrapeptide chain structure formed by joining two identical heavy chains and two identical light chains via interchain disulfide bonds.

The engineered antibodies or antigen binding fragments of the disclosure can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site of the Fc region. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified using conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing conditioned buffer. Non-specifically bound components are washed away. The bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

"Buffering agent" refers to a buffering agent that is resistant to pH changes by the action of its acid-base conjugate components. Examples of buffers to control the pH in the appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine and other organic acid buffers.

A "histidine salt buffer" is a buffer that contains histidine ions. Examples of histidine salt buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate and like buffers, preferably histidine-acetate buffers, which are formulated with histidine and acetic acid, and histidine hydrochloride buffers which are formulated with histidine and hydrochloric acid.

A "citrate buffer" is a buffer that includes citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate.

A "succinate buffer" is a buffer that includes succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. The preferred succinate buffer is succinic acid-sodium succinate. Illustratively, the sodium succinate-succinate may be formulated from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate salt.

A "phosphate buffer" is a buffer that includes phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like. The preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

An "acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine acetate, acetic acid-potassium acetate, calcium acetate, acetic acid-magnesium acetate, and the like. The preferred acetate buffer is acetic acid-sodium acetate.

"Pharmaceutical composition" means a mixture comprising one or more of the antibody drug conjugates described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to maintain the stability of the active ingredients of the antibody, promote the administration to organisms, and facilitate the absorption of the active ingredients so as to exert biological activity.

In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

The pharmaceutical compositions described in this disclosure are in the form of solutions, wherein the solvent is water unless otherwise specified.

By "lyophilized formulation" is meant a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a vacuum freeze-drying step of a liquid or solution formulation.

The terms "about" and "approximately" as used herein mean that the value is within an acceptable error range for the particular value being determined by one of ordinary skill in the art, the value depending in part on how the measurement or determination is made (i.e., the limits of the measurement system). For example, "about" in each implementation in the art may mean within 1 or exceeding a standard deviation of 1. Or "about" or "substantially comprising" may mean a range of up to 20%. Furthermore, the term may mean at most one order of magnitude or at most 5 times the value, especially for biological systems or processes. Unless otherwise indicated, when a particular value is found in the present disclosure and claims, the meaning of "about" or "consisting essentially of" should be assumed to be within the acceptable error range for that particular value.

The pharmaceutical compositions of the present disclosure are capable of achieving a stabilizing effect in which the antibody drug conjugate substantially retains its physical and/or chemical stability and/or biological activity after storage, preferably the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The shelf life is generally selected based on the predetermined shelf life of the pharmaceutical composition. There are a number of analytical techniques for measuring protein stability that measure stability after storage at a selected temperature for a selected period of time.

Stable formulations are those in which no significant change is observed when stored at refrigeration temperatures (2-8 ℃) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. In addition, stable liquid formulations include those that exhibit desirable characteristics after storage at temperatures including 25 ℃ for periods including 1 month, 3 months, and 6 months. Typical examples of stability are aggregation or degradation of typically no more than about 10%, preferably no more than about 5% of the antibody monomer as measured by SEC-HPLC. The formulations were pale yellow, nearly colorless clear liquids or colorless, or clear to slightly milky in color by visual analysis. The concentration, pH and osmolality of the formulation have a variation of no more than + -10%. A reduction of no more than about 10%, preferably no more than about 5%, is generally observed. Usually no more than about 10%, preferably no more than about 5% of aggregates are formed.

An antibody drug conjugate "retains its physical stability" in a drug formulation if it does not exhibit significant increases in aggregation, precipitation and/or denaturation after visual inspection of color and/or clarity, or as measured by UV light scattering, size Exclusion Chromatography (SEC) and Dynamic Light Scattering (DLS). The change in protein conformation can be assessed by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

An antibody drug conjugate "retains its chemical stability" in a pharmaceutical formulation if the antibody does not exhibit a significant chemical change. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often alter the chemical structure of proteins include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (assessed by methods such as peptide spectroscopy in combination with mass spectrometry or MALDI/TOF/MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide spectroscopy, isoaspartic acid measurement, etc.), and isomerization (assessed by measuring isoaspartic acid content, peptide spectroscopy, etc.).

An antibody drug conjugate "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody drug conjugate at a given time is within a predetermined range of biological activity exhibited when the pharmaceutical formulation is prepared.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may be, but need not be, present.

"Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable when bound to carbon atoms having unsaturated (e.g., olefinic) bonds.

The preparation of the conventional pharmaceutical composition is shown in Chinese pharmacopoeia.

The term "carrier" as used in this disclosure refers to a system that alters the manner and distribution of a drug into the body, controls the release rate of the drug, and delivers the drug to a targeted organ. The drug carrier release and targeting system can reduce drug degradation and loss, reduce side effects and improve bioavailability. For example, a polymer surfactant which can be used as a carrier can be self-assembled due to the unique amphiphilic structure of the polymer surfactant to form various forms of aggregates, and preferable examples are micelles, microemulsions, gels, liquid crystals, vesicles and the like. These aggregates have the ability to entrap drug molecules while having good permeability to membranes and can be used as good drug carriers.

"Administration" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid refers to the contact of an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration" and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

By "treating" is meant administering to a patient an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present disclosure, that has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease symptom can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses to assess the severity or progression of the symptom. While embodiments of the present disclosure (e.g., therapeutic methods or articles of manufacture) may be ineffective in alleviating each target disease symptom, it should be determined according to any statistical test method known in the art, such as Student t-test, chi-square test, U-test according to Mann and Whitney, kruskal-Wallis test (H test), jonckheere-Terpstra test, and Wilcoxon test, that the target disease symptom should be alleviated in a statistically significant number of patients.

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical disorder. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending upon, for example, the condition to be treated, the general health of the patient, the route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

"Displacement" refers to the displacement of a solvent system that solubilizes an antibody protein, e.g., a physically manipulated high salt or hypertonic solvent system containing an antibody protein using a buffer system of a stable formulation, such that the antibody protein is present in the stable formulation. The physical means of operation include, but are not limited to, ultrafiltration, dialysis or reconstitution after centrifugation.

Detailed Description

The present disclosure is further described below in connection with the examples, which are not intended to limit the scope of the present disclosure. The experimental methods for which specific conditions are not specified in the examples of the present disclosure are generally conducted under conventional conditions, for example, under conditions recommended by the manufacturer of raw materials or commodities, as described in the antibody technical laboratory Manual, molecular cloning Manual, published in Cold spring harbor laboratory. The reagents of specific origin are not noted and are commercially available conventional reagents.

Biological evaluation

1. Preparation of anti-B7H 3-ADC preparation ADC-1

The anti-B7H 3-ADC structure of the present disclosure is as follows:

Dilute the bare antigen solution h1702DS (6.43L, 1.39 mmol) with pH 5.0 acetic acid-sodium acetate buffer (0.98L, 30 mM), then sequentially add EDTA solution (0.57L, 29 mmol), pH 8.2 histidine-acetic acid-Tris solution (1.47L, 20 mM) and Tris solution (0.2L, 1M), add Tris (2-carboxyethyl) phosphine (TCEP) solution (1.78L, 3.06 mmol), react for 2 hours at 12℃after mixing, and adjust its pH to 6.0 with acetic acid after the reaction.

0.57L of DMSO solvent was added to the reaction solution, and then a solution of compound 5-A (5-A of example 5 of the specification of WO2020063673A1, which was dissolved in DMSO,6.58g,6.12 mmol) was added dropwise to the reaction solution, and after thoroughly mixing, the mixture was reacted at 12℃for 1 hour. After the reaction, acetic acid was added to adjust the pH of the reaction mixture to 5.0. The reaction solution was purified by cation chromatography (column packing Capto S impAct, eluent 50mM acetic acid-sodium acetate+240 mM NaCl, pH 5.5) and then subjected to ultrafiltration to obtain an exemplary product ADC-1 of the B7H3-ADC antibody drug conjugate of the present disclosure.

DAR values were measured by RP-HPLC and n was 4.1.

2. Formulations

The equipment used in the preparation and detection processes and the result calculation method are as follows:

1) SEC size exclusion chromatography:

A method for analyzing the separation of solutes according to the relative relationship between the pore size of the gel pores and the coil size of the polymer sample molecules.

SEC% (SEC monomer content percentage) =a monomer/atotal 100% (a monomer is the peak area of the main peak monomer in the sample, atotal is the sum of all peak areas.)

SEC measuring instrument Agilent 1260, column water, XBrigeSEC(300×7.8mm3.5μm)

2) CE capillary gel electrophoresis:

a method of electrophoresis by moving the gel into a capillary as a supporting medium and separating according to the molecular weight of the sample under a certain voltage.

Reduction CE purity percentage = a main peak/a total 100% (a main peak is peak area of light chain main peak + heavy chain main peak in sample, a total is sum of all peak areas).

CE measuring instrument Beckman model plus800

3) Turbidity measurement:

The degree to which light is blocked when passing through the water layer indicates the ability of the water layer to scatter and absorb light, not only in relation to the suspended matter content, but also in relation to the particle composition, size, shape and reflective properties of the surface. By comparing absorbance values at the same concentration and at the same wavelength (near ultraviolet and visible wavelength regions) for the same protein sample, a greater absorbance value indicates a greater turbidity and a more pronounced tendency for protein molecules to aggregate in the sample. The measuring instrument is a multifunctional enzyme-labeled instrument (Molecular Devices M), and the same volume of sample is added into a 96-well plate to read absorbance values.

4) Osmotic pressure measurement:

the freezing point method is used for measuring the osmotic pressure, the freezing point of the solution is measured by adopting a high-sensitivity temperature sensing element based on the direct proportional relation between the freezing point depression value and the molar concentration of the solution, and the solution is converted into the osmotic pressure through electric quantity. Instrument manufacturer Luo Ze Loser, model OM815.

5) Protein concentration determination:

The concentration of the antibody drug conjugate in the present disclosure is in terms of the concentration of the protein, i.e., in terms of the concentration of the antibody moiety in the antibody drug conjugate.

Because the toxin in the antibody drug conjugate absorbs at 280nm, the protein characteristic absorption wavelength, and the toxin also absorbs at 370nm, the protein concentration was calculated as A _280nm＝(C_drug×E_drug-280+C_mAb×E_mAb-280)×lA_370nm＝C_drug×E_drug-370 ×l using the formula

Taking out

Namely:

Wherein, A _280nm is the average value of absorbance of a single sample of the test solution at a wavelength of 280nm when the optical path is 1 cm;

A _370nm average value of absorbance of single sample of test solution at 370nm wavelength when optical path is 1 cm;

e _mAb-280 the mass extinction coefficient of the protein at 280nm wavelength is 1.532g ^-1cm^-1 L;

E _drug-280, mass extinction coefficient of toxin at 280nm wavelength, 5.17g ^-1cm^-1 L;

E _drug-370 the mass extinction coefficient of the toxin at 370nm wavelength, 17.89g ^-1cm^-1 L;

R is the ratio of toxin extinction coefficient between 370nm and 280nm, which is 3.46;

C _mAb concentration of protein, mg/mL;

l optical path length, cm (here optical path is 1 cm).

If the test solution is diluted, the protein concentration is C (mg/mL) =C _mAb ×N, N is the dilution multiple.

The protein concentration measuring instrument is an ultraviolet-visible spectrophotometer, and the model is Nano Drop One.

6) Drug loading calculation (reverse phase high performance liquid chromatography, RP-HPLC)

1. Reagents and instrumentation:

HPLC system, waters H-Class ultra-high performance liquid chromatograph UPLC system

Detector TUV detector (measurement wavelength: 280 nm)

BioResolve RP mAb Polyphenyl chromatographic columns (2.7 μm 4.6. Times.150 mm)

2. Detection conditions:

Column temperature 80 DEG C

Flow Rate 1.0 mL/min

Mobile phase a 0.1% formic acid+0.025% trifluoroacetic acid (TFA) in water.

Mobile phase B0.1% fa+0.025% trifluoroacetic acid (TFA) acetonitrile solution.

Gradient procedure 27.0% B-45.0% B (0.00 min-12.00 min), 45.0% B-80.0% B (12.00 min-13.00 min), 80.0% B-80.0% B (13.00 min-15.00 min), 27.0% B-27.0% B (15.04 min-20.00 min),

The amount of the injected sample was 5.0. Mu.L

3. Data analysis

The hydrophobicity of the drug-binding light chain (1 drug-binding light chain: L ₁) and the drug-binding heavy chain (1 drug-binding heavy chain: H ₁, 2 drug-binding heavy chain: H ₂, 3 drug-binding heavy chain: H ₃, 4 drug-binding heavy chain: H ₄) increased in direct proportion to the number of drug-binding thereof, and the retention time was prolonged, as compared to the drug-free antibody light chain (L ₀) and the antibody heavy chain (H ₀). Thus, separate elution may be performed in the order of L ₀、L₁、H₀、H₁、H₂、H₃ and H ₄. As a result of comparing the retention times between L ₀ and H ₀, a detected peak was assigned to either of L ₀、L₁、H₀、H₁、H₂、H₃ and H ₄.

Since the drug linker absorbs UV, the resulting peak area is corrected according to the number of bound drugs using the molar absorption coefficients of the light chain, heavy chain and drug linker according to the following expression. The calculation formula is as follows:

light chain (epsilon LC-280)/(epsilon LC-280+ number of linked drugs epsilon drug-280)

Heavy chain (epsilon HC-280)/(epsilon HC-280+ number of linked drugs epsilon drug-280)

Remarks epsilon LC-280: molar extinction coefficient of the light chain at 280 nm;

Epsilon HC-28. Molar extinction coefficient of heavy chain at 280 nm;

Epsilon drug-280. Molar extinction coefficient of toxin at 280 nm.

Table 1 reverse phase chromatography drug loading calculation table

Remarks LC corrected peak area sum = L ₀ corrected peak area + L ₁ corrected peak area

The sum of HC corrected peak areas = H ₀ corrected peak area + H ₁ corrected peak area + H ₂ corrected peak area + H ₃ corrected peak area + H ₄ corrected peak area, and the drug loading of the ADC drug was calculated as drug loading n = 2 x sigma (number of linked drugs x corrected peak area percentage)/100

Example 1 anti-B7H 3-ADC antibody preparation buffer System and pH screening

An anti-B7H 3-ADC preparation containing 60mg/mL sucrose and 0.2mg/mL polysorbate 80 (abbreviated as PS 80) was prepared with a protein concentration of ADC-1 of 20mg/mL using the following buffers:

1) 10mM citric acid-sodium Citrate (CA) pH5.0;

2) 10mM succinic acid-succinic acid sodium salt (abbreviated as SA), pH5.0;

3)10mM SA,pH 5.5;

4) 10mM histidine-hydrochloride (His), pH5.5;

5)10mM His,pH6.0;

6)10mM His,pH6.5;

7) 10mM histidine-acetate (His-AA for short), pH5.0;

8)10mM His-AA,pH5.5;

9) 10mM disodium hydrogen phosphate-sodium dihydrogen Phosphate (PB) pH6.5.

And filtering, filling, plugging and capping the prepared preparation. The stability of the samples under forced degradation conditions (40 ℃ CM1, i.e. 40 ℃ C. For 1 month at high temperature; 300rpm shaking D10, i.e. shaking for 10 days) was examined, and the stability of the formulations was examined using appearance, SEC and reducing CE-SDS as evaluation indexes. The experimental results are shown in Table 2.

Under the forced degradation condition of shaking D10, the appearance of the protein preparation of the SA and His-AA buffer system is superior to that of the CA, his and PB systems, no obvious difference exists between the purity items of the SA and His-AA, CA, his buffer system and is superior to that of the PB system, and under the forced degradation condition of 40 DEG CM1, the appearance of the protein preparation of the SA and His-AA buffer system is superior to that of the CA and PB systems. In combination, SA and His-AA are preferred for subsequent screening of the formulation as B7H3-ADC buffer systems, preferably at pH 5.0-5.5, more preferably at pH 5.5.

Table 2 pH and buffer System screening test results

Remarks D0 indicates at the beginning of the experiment, "M" indicates a month, e.g., M1 indicates a month

Example 2 surfactant screening in anti-B7H 3-ADC antibody formulations

Using a 10mM SA, pH5.0 buffer system, anti-B7H 3-ADC formulations containing 60mg/mL sucrose, different classes and concentrations of surfactant with a protein concentration of ADC-1 of 20mg/mL were formulated. The method comprises the following steps:

1) 0.2mg/mL polysorbate 80 (PS 80);

2)0.4mg/mL PS80;

3) 0.2mg/mL polysorbate 20 (abbreviated as PS 20).

And filtering, filling, plugging and capping the prepared preparation. The samples were subjected to shaking (25 ℃,300rpm,6 days), freeze thawing (-35 ℃ C. -2-8 ℃ C.) for 5 cycles and high temperature (40 ℃ CM 1) with appearance, SEC, and reduction R-CE as evaluation indexes, and stability of the formulations was examined. The experimental results are shown in Table 3.

Experimental results show that under the placement condition, there is no significant difference in appearance and purity terms between the 0.2mg/mL PS80, 0.4mg/mL PS80 and 0.2mg/mL PS20 groups.

Table 3 surfactant screening test results in formulations

Note that "D" in the table means day, e.g., D6 means 6 days, D0 means at the beginning of the experiment, "M" means month, e.g., M1 means 1 month, and "FT 5Cycle" means freeze thawing for 5 cycles.

Example 3 screening of anti-B7H 3-ADC antibody preparation sugar

An anti-B7H 3-ADC formulation containing 0.2mg/mL polysorbate 80 and having a protein concentration of 20mg/mL for ADC-1 was formulated using a 10mM SA, pH5.0 buffer system. The different sugars were as follows:

1) 60mg/mL sucrose;

2) Trehalose at 60 mg/mL;

3) 50mg/mL mannitol.

And filtering, filling, plugging and capping the prepared preparation. The stability of the sample under forced degradation conditions (shaking D6, i.e. shaking for 6 days; FT 5Cycle, i.e. freeze thawing for 5 cycles at-35 ℃ C. -2 to 8 ℃ C.) was examined, and the stability of the formulation was examined using SEC as an evaluation index. The experimental results are shown in Table 4.

Experimental data show that SEC purity terms for sucrose and trehalose groups are superior to mannitol groups under shaking D6 and FT5C forced degradation conditions. Subsequently, an anti-B7H 3-ADC antibody preparation containing 20mg/mL of antibody, 60mg/mL of sucrose, and 0.2mg/mL of PS80 was prepared in a buffer solution of 10mM SA pH5.0, the preparation sample was lyophilized, the appearance bottom collapsed after lyophilization, and the optimization was continued, so that the sucrose was reduced from 60mg/mL to 40mg/mL.

Table 4 results of adjuvant screening experiments

Remarks in the table, "D" indicates day and D0 indicates the start of the experiment.

Example 4 screening of osmolality regulator in anti-B7H 3-ADC antibody formulations

An anti-B7H 3-ADC formulation containing 40mg/mL sucrose, 0.2mg/mL PS80, protein concentration of ADC-1 was formulated using a 10mM SA, pH5.5 buffer system. The osmotic pressure of the preparation is controlled within the isotonic range (270-330 mosm) by improving the ionic strength and screening the osmotic pressure regulator.

Using 30mM SA, pH5.5 buffer system, anti-B7H 3-ADC formulations containing 40mg/mL sucrose, 0.2mg/mL PS80, different types of isotonic osmotic regulator with protein concentration of ADC-1 of 20mg/mL were formulated. The method comprises the following steps:

1) 58mM arginine hydrochloride;

2) 7.6mg/mL glycine;

the prepared preparation is filtered, filled, half plugged and freeze-dried. The lyophilization procedure is as follows in table 5:

Table 5 lyophilization procedure

The stability of the lyophilized sample under forced degradation conditions (40 ℃ CD23, i.e. at 40 ℃ for 23 days) was examined, and the stability of the formulation was examined using appearance, SEC and reduced R-CE as evaluation indexes. The experimental results are shown in Table 6.

Under the condition of forced degradation of 40 DEG CD23, the purity items among the groups have no obvious difference, and the appearance of the glycine group is obviously superior to that of the arginine hydrochloride group. When the osmotic pressure of a preparation containing 40mg/mL sucrose and 7.6mg/mL glycine in the sample is about 300mOsm, a glycine concentration of 7.6mg/mL is preferred. The osmotic pressure regulator glycine is used, and when the concentration is 5.3mg/mL-9.8mg/mL, the osmotic pressure of the preparation is controlled within the isotonic range.

Table 6 results of osmolality regulator screening experiments in formulations

Remarks in the table "D" means day, e.g. D23 means 23 days.

Example 5 screening of protein concentration in anti-B7H 3-ADC antibody preparations

Different protein concentrations of anti-B7H 3-ADC formulations containing 40mg/mL sucrose, 0.2mg/mL PS80,7.6mg/mL glycine were formulated using 30mM SA, pH5.5 buffer system. The method comprises the following steps:

1)20mg/mL ADC-1

2)50mg/mL ADC-1

3)60mg/mL ADC-1

4)70mg/mL ADC-1

And filtering, filling, plugging and capping the prepared preparation. The samples were subjected to shaking (25 ℃,300rpm,6 days), freeze thawing (-35 ℃ C. -2-8 ℃ C.) for 5 cycles and high temperature (40 ℃ CM 1) with appearance, SEC, DAR values as evaluation indicators, and formulation stability was examined. The experimental results are shown in Table 7.

Experimental results show that under the placement condition, there is no significant difference in appearance, purity terms and toxin load between the 20mg/mL ADC-1, 50mg/mL ADC-1, 60mg/mL ADC-1 and 70mg/mL ADC-1 groups.

Table 7 surfactant screening test results in formulations

Note that "D" in the table indicates day, D0 indicates the beginning of the experiment, "M" indicates month, e.g., M1 indicates 1 month, and "FT 5Cycle" indicates 5 cycles of freeze thawing.

Example 6 stability test of anti-B7H 3-ADC antibody preparation

An anti-B7H 3-ADC formulation was prepared containing ADC-1 at a protein concentration of 20mg/mL,30mM succinic acid-succinic acid sodium Salt (SA) pH5.5,40mg/mL sucrose, 7.6mg/mL glycine and 0.2mg/mL PS 80.

Filtering the prepared preparation, filling, adding a plug, and capping. The stability of the preparation is inspected by taking appearance, SEC and reduction R-CE as evaluation indexes under forced degradation conditions (-35 ℃ C. To 2 ℃ C. To 8 ℃ C. Freeze thawing for 5 cycles, or shaking for 8 days) and long-term standing (2 to 8 ℃ C. For 16 days) of the sample. The experimental results are shown in Table 8.

Experimental results show that the product is clear under all forced degradation conditions from the appearance, has no obvious difference in purity items, and has good stability.

TABLE 8 results of stability experiments on formulations

Remarks in the table, "D" indicates day, D0 indicates start of experiment, FT5C indicates freeze-thaw 5 cycles.

EXAMPLE 7B 7H3-ADC formulation prescription optimization experiments

Optimization experiment one:

anti-B7H 3-ADC formulations containing 7.6mg/mL glycine, 40mg/mL sucrose were subjected to DOE design of experiments with the pH of 30mM succinic acid-succinic acid sodium Salt (SA), protein concentration of ADC-1 and PS80 concentration as variables. DOE, experimental factors and levels were set to pH 5-6, protein concentration of ADC-1 10-30 mg/mL, PS80 concentration 0.1-0.3 mg/mL, and a series of prescriptions were designed (see Table 9). The freeze-dried sample is subjected to forced degradation experiment at 40 ℃ for one month, and the appearance and SEC are taken as evaluation indexes, and the results are shown in Table 9.

The results show that each prescription was clear in appearance with no significant differences between the SEC groups at 40℃for one month. Therefore, the B7H3-ADC sample has good stability in the ranges of pH value of 5-6, protein concentration of ADC-1 of 10-30 mg/mL and PS80 concentration of 0.1-0.3 mg/mL.

Table 9 DOE prescription screening Experimental prescription design

Table 10 DOE screening test results

Remarks in the table, "D" indicates day, D0 indicates the start of the experiment, "M" indicates month, e.g., M1 indicates one month.

Optimizing experiment II:

A series of prescriptions were designed based on 30mM SA, succinic acid-succinic acid sodium salt buffer pH5.5, protein concentration of ADC-1 50mg/mL, sucrose 40mg/mL, glycine 7.6mg/mL and PS80 0.2mg/mL anti-B7H 3-ADC preparation, with the buffer concentration, pH, sucrose concentration, glycine concentration, PS80 concentration, protein concentration of ADC-1 as variables (see Table 11). The freeze-dried sample was subjected to forced degradation test at 40 ℃ for one month, and the appearance, SEC purity and DAR value were used as evaluation indexes, and the results are shown in table 12.

The results showed that each prescription was clear in appearance with no significant differences between the purity term SEC and DAR toxin loading groups when left for one month at 40 ℃ high temperature. Therefore, the B7H3-ADC sample has good stability in the ranges of SA concentration of 20-30 mM, pH value of 5.0-5.5, protein concentration of ADC-1 of 20-70 mg/mL, sucrose concentration of 40-80 mg/mL and PS80 concentration of 0.2-0.8 mg/mL.

Table 11 DOE prescription screening Experimental prescription design

Table 12 DOE screening test results

Note that "D" in the table indicates day, "W" indicates week, D0 indicates the beginning of the experiment, and W4 indicates four weeks.

Example 8 lyophilization of anti-B7H 3-ADC antibody preparation

An anti-B7H 3-ADC antibody preparation containing ADC-1 at a protein concentration of 20mg/mL,40mg/mL sucrose, 7.6mg/mL glycine and 0.2mg/mL PS80 was prepared in30 mM SA, pH5.5 succinic acid-succinic acid sodium salt buffer. The preparation samples were lyophilized by pre-lyophilization, primary drying and secondary drying procedures, see table 13. After the lyophilization procedure was completed, vacuum stoppered. The samples before and after freeze-drying are evaluated by taking appearance, SEC and R-CE as evaluation indexes, and the results are shown in the results in Table 14, wherein the reconstituted solution can keep good performance of the liquid preparation.

Table 13 lyophilization procedure

Table 14 results of experiments before and after lyophilization

Claims (17)

1. A pharmaceutical composition comprising an antibody drug conjugate and a buffer, wherein the antibody drug conjugate has the structure shown below:

   Wherein:

   h1702DS is an anti-B7H 3 antibody comprising a heavy chain as shown in SEQ ID NO. 1 and a light chain as shown in SEQ ID NO. 2;

   n is 1 to 10, preferably 1 to 8, more preferably 3 to 5, more preferably about 4;

   The buffer of the pharmaceutical composition is succinate buffer, histidine buffer, preferably succinic acid-succinic acid sodium salt, histidine-acetate.
2. The pharmaceutical composition of claim 1, wherein the buffer is at a concentration of about 10mM to about 50mM, preferably about 20mM to about 40mM, more preferably about 30mM.
3. The pharmaceutical composition according to claim 1 or 2, wherein the pH of the pharmaceutical composition is about 4.5 to 6.5, preferably about 5.0 to 6.0, more preferably about 5.5 to 5.6.
4. A pharmaceutical composition according to any one of claims 1 to 3, further comprising a surfactant, preferably polysorbate, more preferably polysorbate 80 or polysorbate 20, most preferably polysorbate 80.
5. The pharmaceutical composition of claim 4, wherein the surfactant concentration is about 0.01mg/mL to about 1.0mg/mL, preferably about 0.2mg/mL to about 0.8mg/mL, more preferably about 0.2mg/mL.
6. The pharmaceutical composition according to any one of claims 1 to 5, further comprising a sugar, preferably the sugar is selected from sucrose, mannitol and trehalose, more preferably the sugar is sucrose or trehalose.
7. The pharmaceutical composition of claim 6, wherein the sugar concentration is about 25mg/mL to about 80mg/mL, preferably about 40mg/mL to about 60mg/mL, more preferably about 40mg/mL.
8. Pharmaceutical composition according to any one of claims 1 to 7, characterized in that it further comprises an amino acid or an amino acid salt selected from glycine and arginine hydrochloride, preferably glycine.
9. The pharmaceutical composition of claim 8, wherein the amino acid or amino acid salt concentration is about 5mg/mL to about 10mg/mL, preferably about 7mg/mL to about 8mg/mL, more preferably about 7.6mg/mL.
10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the antibody drug conjugate concentration is about 10mg/mL to about 70mg/mL, preferably about 20mg/mL to about 50mg/mL, more preferably about 20mg/mL to about 30mg/mL or about 30mg/mL to about 50mg/mL, even more preferably about 20mg/mL or about 30mg/mL or about 50mg/mL, based on protein concentration.
11. A pharmaceutical composition according to any one of claims 1 to 10, comprising the following components:

    (a) from about 20mg/mL to about 70mg/mL of the antibody drug conjugate, based on protein concentration, (b) from about 0.2mg/mL to about 0.8mg/mL of polysorbate, (c) from about 25mg/mL to about 80mg/mL of sugar, (d) from about 7mg/mL to about 8mg/mL glycine, and (e) from about 10mM to about 40mM succinate buffer, the pH of the composition being from about 5.0-6.0;

    Preferably, the pharmaceutical composition comprises the following components:

    (a) from about 20mg/mL to about 50mg/mL of the antibody drug conjugate, based on protein concentration, (b) from about 0.2mg/mL to about 0.8mg/mL of polysorbate 80, (c) from about 40mg/mL to about 80mg/mL of sucrose, (d) from about 7.6mg/mL of glycine, and (e) from about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being from about 5.5 to 5.6;

    more preferably, the pharmaceutical composition comprises the following components:

    (a) from about 20mg/mL to about 30mg/mL of the antibody drug conjugate, based on protein concentration, (b) about 0.2mg/mL polysorbate 80, (c) about 40mg/mL sucrose, (d) about 7.6mg/mL glycine, and (e) about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being about 5.5-5.6;

    (a) from about 30mg/mL to about 50mg/mL of the antibody drug conjugate, based on protein concentration, (b) about 0.2mg/mL of polysorbate 80, (c) about 40mg/mL of sucrose, (d) about 7.6mg/mL glycine, and (e) about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being about 5.5-5.6.
12. A lyophilized formulation comprising an antibody drug conjugate, wherein the lyophilized formulation, upon reconstitution, forms a pharmaceutical composition according to any one of claims 1 to 11.
13. A method of preparing a lyophilized formulation of an antibody-containing drug conjugate, comprising the step of lyophilizing the pharmaceutical composition of any one of claims 1 to 11.
14. A lyophilized formulation comprising an antibody drug conjugate, characterized in that the lyophilized formulation is obtained by freeze-drying a pharmaceutical composition according to any one of claims 1 to 11.
15. A reconstituted solution comprising an antibody drug conjugate, wherein the reconstituted solution is prepared by reconstitution of the lyophilized formulation of claim 12 or 14, and the reconstituted solution comprises the following components:

    (a) from about 20mg/mL to about 70mg/mL of the antibody drug conjugate, based on protein concentration, (b) from about 0.2mg/mL to about 0.8mg/mL of polysorbate, (c) from about 25mg/mL to about 80mg/mL of sugar, (d) from about 7mg/mL to about 8mg/mL glycine, and (e) from about 10mM to about 40mM succinate buffer, the pH of the composition being from about 5.0-6.0;

    preferably, the redissolved solution comprises the following components:

    (a) from about 20mg/mL to about 50mg/mL of the antibody drug conjugate, based on protein concentration, (b) from about 0.2mg/mL to about 0.8mg/mL of polysorbate 80, (c) from about 40mg/mL to about 80mg/mL of sucrose, (d) from about 7.6mg/mL of glycine, and (e) from about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being from about 5.5 to 5.6;

    More preferably, the redissolved solution comprises the following components:

    (a) from about 20mg/mL to about 30mg/mL of the antibody drug conjugate, based on protein concentration, (b) about 0.2mg/mL polysorbate 80, (c) about 40mg/mL sucrose, (d) about 7.6mg/mL glycine, and (e) about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being about 5.5-5.6;

    (a) from about 30mg/mL to about 50mg/mL of the antibody drug conjugate, based on protein concentration, (b) about 0.2mg/mL of polysorbate 80, (c) about 40mg/mL of sucrose, (d) about 7.6mg/mL glycine, and (e) about 30mM succinic acid-succinic acid sodium salt, the pH of the composition being about 5.5-5.6.
16. An article of manufacture comprising a container containing the pharmaceutical composition of any one of claims 1 to 11, the lyophilized formulation of claim 12 or 14, or the reconstituted solution of claim 15.
17. Use of a pharmaceutical composition according to any one of claims 1 to 11, a lyophilized formulation according to claim 12 or 14, a reconstituted solution according to claim 15 or a preparation according to claim 16 for the manufacture of a medicament for the treatment of a disease, preferably a cancer associated with B7H3 expression, more preferably selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, renal cancer, urinary tract cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.